Background: Metastasis causes most deaths from cancer. In a number of studies, high expressions of the MARCKS protein (Myristoylated Alanine-Rich C Kinase Substrate, a 332-amino acid protein known to play a key role in normal cell motion) in cancer cells have been associated with higher rates of metastasis in breast, lung, prostate, brain, skin and other neoplasias. Recently, Chen et al. showed that the 24-amino acid N-terminal sequence of MARCKS, called the MANS peptide, blocked metastasis of lung cancer cells orthotopically injected into mouse lungs (http://www.ncbi.nlm.nih.gov/pubmed/23955080). Unfortunately, for various reasons, the MANS peptide is not practical as an anti-cancer drug candidate. We have identified shorter and far more soluble N-terminal peptide inhibitors of MARCKS that appear to block migration of cancer cells in vitro and metastasis in mice in-vivo at lower concentrations than the MANS peptide, and when administered either IP or as an inhaled aerosol.
Methods: For the in vitro cancer cell migration assays, 1 X 105 of three separate human lung cancer cell lines (A549, PC-9, and CL1-5) were cultured in Transwell ® plates (24-well, 8-um pore size). Either PBS (control), MANS, or 3 different N-terminal peptide inhibitor analogs of MANS; BIO-11006, BIO-11002, and BIO-10901 were incubated with the cells for 12 hrs, and cells that migrated from the upper to lower surface of the chambers were stained with hematoxylin and counted. For the in vivo metastasis evaluations, PC-9 cells were orthotopically-injected into the lungs of NOD-SCID mice with or without preincubation with either MANS or BIO-11006, and animals were given MANS or BIO-11006 IP after 7 days and every 3 days up until day 25, when they were harvested and examined. Another group of orthotopically injected animals received treatment with BIO-11006 as an inhaled aerosol starting at day 4 and every day thereafter until day 25. Animals were euthanized at day 26; locations and magnitudes of systemic metastases were assessed macroscopically and microscopically.
Results: All of the N-terminal peptide inhibitors showed concentration - dependent attenuation of in vitro migration of each of the cancer cell lines, with an inhibition of close to 75% at the highest concentration (100μM). The smaller MANS analogs appeared to be the most effective at the lower concentrations. IP injection of MANS or BIO-11006 dramatically inhibited metastasis of the primary tumor to other sites in the lung, heart and diaphragm, with BIO-11006 showing potent inhibition of metastasis even at a concentration an order of magnitude lower than MANS. Administration of 50 uM BIO-11006daily via inhaled aerosol had the same inhibitory effect on metastasis as IP injection of 50 uM every three days.
Conclusion: N-terminal peptide inhibitors of MARCKS suitable for development as potential new anti-cancer drugs show substantial promise in inhibiting metastasis of lung cancer cells both in vitro and in vivo.
Citation Format: Walker A. Long, Indu Parikh, Qi Yen, Shijing Fang, Anne L. Crews, Kenneth B. Adler. MARCKS protein inhibitors attenuate cancer cell migration/metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2014-4042